Lactobacillus is a genus of bacteria in the family Lactobacteriacae, which are found in the intestinal tracts of mammals, on green plants, in milk and fermented foods. Lactic acid is produced by these bacteria via carbohydrate fermentation; thus, lactobacilli have been introduced into the manufacture of food products, such as yogurt and cheese, to enhance their quality and stability. In addition, lactobacilli form an important part of the intestinal microbial flora in humans and other animals. In humans, lactobacilli are present in the mouth, lower intestine and vagina.
Other types of bacteria, for example Escherichia coli (hereafter E. coli), are also present in animals. Pathogenic strains can cause serious intestinal problems such as diarrhea and gastroenteritis. A proper balance of the microbial populations in the intestinal tract of animals is believed to be important to provide resistance to such diseases. Conditions such as stress can alter the balance of microbes in the intestinal tracts of humans and other animals making them more susceptible to disease. This knowledge of the importance of maintaining a proper balance of microorganisms has led to the development of therapy designed to maintain the proper balance. Such therapy has included the use of strains of L. acidophilus in dried form, administered orally, for promoting L. acidophilus colonization in human intestines. For a review see W. E. Sandine et al. J. Food Protection 42:259-262 (1979). Moreover, L. acidophilus has shown some effectiveness in inhibiting E. coli in infants suffering from diarrhea.
L. acidophilus has also been used in animal feed in an attempt to restore and stabilize the intestinal microbial balance. In some cases, animals such as pigs showed improved growth and exhibited a decreased population of E. coli when administered lactobacilli. Two patents (U.S. Pat. No. 3,343,962 and U.K. Pat. No. 1,134,206) disclose methods of preparation of certain lactobacilli for use in animal feed supplements.
Unfortunately, in field studies outside of the laboratory, prior preparations using known strains of Lactobacillus have proven ineffective, in part a) because sufficiently high numbers of viable microorganisms have not been present in the feed preparations and/or b) the organisms were unable to successfully colonize the subject being treated. In such studies it would be desirable to identify and enumerate lactobacilli in feed preparations and intestinal and fecal contents but such laboratory measurements are complicated by the large number of environmental microorganisms commonly encountered in these field samples.
It is thought that adhesion is a primary event in bacterial colonization of a particular habitat, such as the intestinal tract. Bacteria are known to adhere to various surfaces, including human and animal cells; see G. W. Jones, "The Attachment of Bacteria to the Surfaces of Animal Cells," in Microbial Interactions (Russing, E.), Chapman and Hall, London (1977), incorporated by reference herein. Attachment appears to be important for establishing and maintaining either the normal- or disease-associated bacterial flora in humans and other animals. For example, bacterial attachment to human mucosal epithelial cells, such as vaginal cells, has been studied and related to possible colonization and invasion by pathogens [R. A. Mardh and L. Westrom, Infection and Immunity, 13, p. 661-666(1976); Chan et al., Infection and Immunity, 47, pp. 84-89 (1985)].
Differences exist between strains of Lactobacillus acidophilus in their ability to survive, initiate and maintain a bacterial population within the intestine due, in part, to differences in the ability of the various strains to adhere to intestinal epithelial cells in different species of animals. These differences may hamper the effectiveness of therapeutically- or prophylactically-administered bacteria. Some lactobacilli strains demonstrate species specificity, such that one strain of L. acidophilus from a chicken source will not adhere to ephithelia of a different species, e.g., a rat, as shown by N. Suegara et al., Infection and Immunity, 12, p. 173-179 (1975); and R. Fuller, J. Applied Bact., 45, p. 389-395 (1978). The stomach and intestinal tract also present physical challenges to the growth and survival of microorganisms such as Lactobacillus. For example, the mucosal clearance mechanisms of the small intestine wash out any organisms that cannot attach to intestinal epithelial cells or multiply fast enough to avoid dilution. Thus, it would be highly desirable to have strains of Lactobacillus with known species specificity as well as routine laboratory assays for determining the effectiveness of the administered bacteria in an animal host.
It is believed that bacteria may attach to animal cells through a variety of mechanisms. In one system, cations may provide a bridge via ionic attraction between surfaces of the bacteria and epithelial cells. This system appears to be nonspecific and is calcium dependent. In another mechanism, the bacteria appear to attach by contacting receptor sites on the epithelial cells. This system is calcium independent and has been found to be species specific, suggesting that the ability to adhere (i.e., to recognize receptors) when mediated by this mehanism is under the genetic control of the bacteria. At least one researcher has identified two subpopulations of human L. acidophilus wherein one population requires calcium to adhere to human fetal epithelial cells and attachment of the other was found to be independent of calcium. One of these strains is capable of interspecific attachment. Such calcium independent attachment has been found in only a few other human bacterial strains [E. G. Kleeman and T. R. Klaenhammer, J. Dairy Sci., 65, pp. 2063-2069 (1982)]. Other mechanisms of bacterial attachment may also be involved, [Sherman et al., Appl. Environ. Microbiol. , 52, p. 302-304 (1986), Fuller et al., Am. J. Clin. Nutr., 27, pp. 1305-1312 (1974)].
A commercially available Lactobacillus feed additive concentrate for cattle, containing L. acidophilus BT1386 (ATCC No. 53545) and sold under the trademark COBACTIN.RTM., (BioTechniques Laboratories, Inc., Redmond, Washington), addressed many of the above problems. Although BT1386 has been highly successful as a commercial feedlot additive, further improvements in certain characteristics of the strain are desirable. For instance, it is difficult to recover, identify, and enumerate lactobacilli of strain BT1386 in animal feed, tissue, feces, etc., because of the large number of background microorganisms present in these samples. Quantitation of L. acidophilus is important to ensure that the microbial inoculum of the feed has been properly prepared and that the animal has been inoculated with a proper dose. Utilizing a marker gene to identify bacteria is routine in the art, e.g., a marker gene confering the ability to utilize a unique carbon source, however, this method is not widely applicable to samples from an animal, or from bacterium-containing food compositions because of the large numbers of bacteria present in these samples and the diversity of carbon sources utilized by the bacteria in them.
The present invention relates to novel strains of L. acidophilus that address the problems of recovery, identification, and enumeration which are described above.